1. Field of the Invention
This invention relates to a rapid integrated system which includes a premixed suspension of a plurality of populations of quantitative fluorescent microbead standards, and analytical software correlated to the microbeads. The system allows analysis of flow cytometer performance with the premixed microbeads.
2. Description of the Related Art
Flow cytometers are used to analyze biological cells and particles present in a fluid sample by intersecting a thin stream of the fluid by an illumination source, usually a laser beam. The resulting forward and right angle scattered and fluorescent light is analyzed with the photomultiplier tubes (PMTs) of the flow cytometer. The fluorescent channels of a flow cytometer which are designated by FL1, FL2, FL3 and so forth, are each set with barrier filters to detect a selected specific dye while filtering out signals from dyes that fluoresce at other wavelengths.
Fluorescent microbeads have been used to calibrate fluorescence instruments such as flow cytometers or fluorescence microscopes so that "positive" cells that fluoresce at a particular wavelength may be distinguished from non-fluorescent cells by differences in fluorescence intensity measurements. Reagents and cell preparation techniques may cause complications in the measurement of fluorescence intensity.
As discussed in U.S. Pat. Nos. 4,714,682 and 4,868,126, for each population group of highly uniform microbeads, the number of molecules of equivalent soluble fluorochrome per (MESF) which is necessary to give rise to the same level of fluorescence intensity as the microbeads is determinable using the following steps: (1) the fluorescence intensity of a suspension of microbeads is determined with a fluorometer as compared to solutions of the free fluorescent dye to determine the number of equivalent soluble dye molecules per unit volume of the microbead suspension; (2) the number of microbeads per unit volume is determined; and (3) the number of equivalent soluble dye molecules per unit volume is divided by the number of microbeads per unit volume of the suspension to yield the equivalent soluble fluorescent dye molecules per microbead in the particular microbead sample. The Molecules of Equivalent Soluble Fluorescence (MESF) value indicates the equivalent number of free fluorochrome molecules in a solution that would have the same emission intensity as the microbead standard. These units are only valid when the excitation and emission spectra of the standards and unknown samples are the same. The MESF per microbead in various microbead samples may then be plotted as a function of fluorescent intensity to calibrate a flow cytometer or fluorescence microscope. The disclosure of this patent and all other patents and patent applications discussed herein is incorporated by reference.
Microbeads having antibodies bound thereto are disclosed in U.S. Pat. No. 4,918,004. A kit of said microbeads contains at least two populations of uniformly sized microbeads in which the number of fluorescently labeled antibodies binding to each microbead in these populations is the same within each population but different from population to population. The antibodies may be directly or indirectly bound to the microbeads, with the phrase "directly or indirectly binding" in reference to microbeads and associated fluorescently labeled antibodies being intended to be broadly construed to encompass covalent bonding of the fluorescently labeled antibody directly to the microbead, as well as microbead systems wherein a linking group e.g., an immunological agent such as a protein or hapten is covalently bonded to the microbead, and the fluorescently labeled antibody is in turn covalently bonded or otherwise bound to this linking group. Thus, for example, the latter indirect binding may be effected by covalently bonding to the microbead a primary nonfluorescent antibody with which a secondary fluorescently labeled antibody is covalently bondable, i.e., the secondary fluorescently labeled antibody being a conjugate of the primary nonfluorescent antibody.
Each of the populations of antibody-containing microbeads is run on a flow cytometer or fluorescence microscope to determine its fluorescence intensity. A calibration plot is constructed of the number of fluorescently labeled antibodies as a function of fluorescence intensity so that a sample's unknown number of fluorescent antibodies may be calculated from its fluorescence intensity.
Many problems arise in flow cytometry with respect to uneven instrument performance from day to day and variations in and losses in microbead fluorescence and in variations due to different reagents, preparations and samples. When the results of flow cytometer use are being used for day-to-day clinical comparisons of a patient or comparisons between samples run on different days, it is critical that variations in instrument performance be recognized so that the results are not erroneously interpreted. It is also important that the technique of standardizing the flow cytometer not cause errors in the observations. When microbeads have lost fluorescence, they no longer can be relied upon for standardization or calibration purposes.
When multiply-labeled samples are run on a flow cytometer and a suspension of multiple microbead populations having different labels is used as a standard, it is often difficult to identify which microbeads are labeled with which dye so that the standard can function effectively for use with the samples.
It is therefore an object of this invention to provide a system comprising fluorescently-labeled microbeads and matched software for flow cytometer use, which provides a comprehensive solution to the problem of obtaining reproducible fluorescence intensity measurements.
It is another object of the invention to provide a microbead-software system which can be used objectively to evaluate and plot instrument performance and operating conditions.
It is a further object of the invention to provide a microbead-software system in which the software automatically analyzes a list mode file of premixed microbeads and generates a linear regression plot and statistical data to assess instrument linearity.
It is a further object of the invention to provide a microbead-software system to determine fluorescence threshold sensitivity.
It is a further object of the invention to provide a microbead-software system that allows unequivocal standardization with multiple fluorescent dyes, in which multiple list mode files may be obtained and processed together.
It is a further object of the invention to provide a microbead-software system to allow creation of a file of daily instrument performance.
It is a further object to provide a system where the life of the microbeads and of the software are correlated with each other.
Further objects of this invention are the rapidity of simplicity, accuracy, objectiveness , and cost-effectiveness of use of the microbead-software system.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.